Suemura Shigeki, Kodama Takahiro, Myojin Yuta, Yamada Ryoko, Shigekawa Minoru, Hikita Hayato, Sakamori Ryotaro, Tatsumi Tomohide, Takehara Tetsuo
Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan.
Cancers (Basel). 2019 Sep 13;11(9):1362. doi: 10.3390/cancers11091362.
Regorafenib is used for hepatocellular carcinoma (HCC), but its response does not last long, partly due to chemoresistance acquisition. We performed a clustered regularly interspaced short palindromic repeats (CRISPR)-based loss-of-function genetic screen and aimed to discover molecules involved in regorafenib resistance in HCC. Xenograft tumors established from Cas9-expressing HCC cells with pooled CRISPR kinome libraries were treated with regorafenib or a vehicle. Sequencing analysis identified 31 genes, with the abundance of multiple guide RNAs increased in regorafenib-treated tumors compared to that in vehicle-treated tumors, including 2 paralogues, LATS2 and LATS1, core components of the Hippo signaling pathway. Notably, all eight designed guide RNAs targeting LATS2 increased in regorafenib-treated tumors, suggesting that LATS2 deletion confers regorafenib resistance in HCC cells. LATS2 knockdown significantly mitigated the cytotoxic and proapoptotic effects of regorafenib on HCC cells. LATS2 inhibition stabilized the Hippo signaling mediator YAP, leading to the upregulation of antiapoptotic Bcl-xL and the multidrug resistance transporter ABCB1. Among 12 hepatoma cell lines, the half maximal inhibitory concentration (IC50) values of regorafenib were positively correlated with any of YAP, Bcl-xL and ABCB1 levels. The inhibition of YAP or Bcl-xL in regorafenib-insensitive HCC cells restored their susceptibility to regorafenib. In conclusion, our screen identified the Hippo signaling pathway as the mediator of regorafenib efficacy in HCC.
瑞戈非尼用于治疗肝细胞癌（HCC），但其疗效维持时间不长，部分原因是出现了化学抗性。我们进行了一项基于成簇规律间隔短回文重复序列（CRISPR）的功能丧失基因筛选，旨在发现与HCC中瑞戈非尼抗性相关的分子。用瑞戈非尼或赋形剂处理由表达Cas9的HCC细胞与汇集的CRISPR激酶组文库建立的异种移植肿瘤。测序分析鉴定出31个基因，与赋形剂处理的肿瘤相比，瑞戈非尼处理的肿瘤中多个导向RNA的丰度增加，包括2个旁系同源物LATS2和LATS1，它们是Hippo信号通路的核心成分。值得注意的是，靶向LATS2的所有8个设计的导向RNA在瑞戈非尼处理的肿瘤中均增加，这表明LATS2缺失赋予HCC细胞瑞戈非尼抗性。LATS2敲低显著减轻了瑞戈非尼对HCC细胞的细胞毒性和促凋亡作用。LATS2抑制使Hippo信号介质YAP稳定，导致抗凋亡蛋白Bcl-xL和多药耐药转运蛋白ABCB1上调。在12种肝癌细胞系中，瑞戈非尼的半数最大抑制浓度（IC50）值与YAP、Bcl-xL和ABCB1水平中的任何一个呈正相关。在对瑞戈非尼不敏感的HCC细胞中抑制YAP或Bcl-xL可恢复其对瑞戈非尼的敏感性。总之，我们的筛选确定Hippo信号通路是瑞戈非尼在HCC中疗效的介质。
